First Report of a Group 16SrIII-B Phytoplasma Associated with Decline of China Tree in Brazil.

China tree (Melia azedarach L.), originally from Asia, is an exotic deciduous species in Brazil and is used as an ornamental shade tree in the southern region of the country. Since 2005, plants displaying yellowing, little leaves, witches' broom, and decline have been observed in the State of Rio Grande do Sul. In the streets and avenues of the capital city of Porto Alegre, there are approximately 173 tree species and China tree (6.57% of all trees) is among the top 10 (80,000 China trees and most are symptomatic). Plants with those symptoms are very distinctive and have been found also in the cities of Livramento, Rio Grande, Santa Maria, and Vacaria, places located in seashore areas, and along highways everywhere in the state. The high incidence seems to be related to drought during the last few years. These symptoms are typical of a disease identified by yellowing or decline of China tree associated with phytoplasma and previously reported in the neighboring countries of Argentina, Paraguay, and Bolivia (2). To demonstrate the presence of phytoplasma in diseased trees and to confirm its identity, total DNA was extracted from China tree leaf midribs collected from 10 symptomatic and three asymptomatic plants. Nested PCR was performed with the P1/P7 primer pair in the primary PCR to amplify a 1.8-kb fragment encompassing the 16S rRNA gene, the 16S-23S spacer region, and the 5' end of the 23S rRNA gene, while the secondary PCR was primed by the R16F2n/R16R2 primer pair to amplify a 1.2-kb fragment of the 16S rRNA gene from the 1.8-kb fragment (3,4). DNA fragments of 1.2 kb amplified from nested PCR were analyzed by restriction fragment length polymorphism with restriction enzymes AluI, HhaI, HpaII, KpnI, MboI, MseI, and RsaI, revealing identical profiles for each amplicon and demonstrating that a phytoplasma belonging to group 16SrIII, subgroup B (16SrIII-B) (1) was associated consistently with all symptomatic plants. BLAST analysis revealed 99% identity among these cloned 1.2-kb sequences and representative sequences of phytoplasmas affiliated with group 16SrIII (GenBank Accession Nos. AY081817 and AF147706). A majority consensus sequence representing the phytoplasma found in China trees was selected and deposited in GenBank (Accession No. FJ404775). These results were confirmed by observation with transmission electron microscopy of pleomorphic bodies 400 to 2,000 nm in diameter in the phloem sieve tubes of all symptomatic trees. No phytoplasma was detected or visualized in asymptomatic samples. These results corroborate those from studies conducted in neighboring countries that demonstrated the association between phytoplasmas of group 16SrIII and decline of China trees (1). In conclusion, the current study revealed that a phytoplasma affiliated with group 16SrIII-B is associated with the decline of China tree in Brazil, a disease previously described based solely on symptoms (2). The incidence and severity of the disease are enough to prevent further use of these trees as landscape plants in southern Brazil. References: (1) J. D. Arneodo et al. J. Phytopathol. 155:70, 2007. (2) M. Dalbosco et al. Fitopatol. Bras. 30(Suppl.):177, 2005. (3) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.